Abstract 3979: Tandem peptides targeting HER2 for delivery of CD44 siRNA into HER2+ breast cancer cells <i>in vitro</i>
Authors: James W. Kalogeros, Audreanna Miserendino, Angela Alexander-Bryant, Brian Booth
Journal: Cancer Research (2023)

Abstract Introduction: This research aims to characterize and evaluate the potential of two novel tandem peptides for their ability to mediate the delivery of CD44 siRNA into HER2+ breast cancer cell lines. HER2+ cancer accounts for 20%-25% of invasive breast cancers and shows overexpression of HER2 protein. The tandem peptides examined consist of P51 or P25 targeting peptide sequences that bind to the HER2 protein, and DIV3W, a fusogenic peptide that allows for siRNA protection, cellular internalization, and endosomal escape. We hypothesize that delivery of tandem peptide will result in the observation of mRNA knockdown of CD44, a reduction of CD44 protein expression, and a decrease in metastatic properties of breast cancer cells. Methods: Western blot analysis was used to determine the basal expression of CD44 in HER2+ breast cancer cell lines, SKBR3 and BT474, and breast epithelial cell line MCF10A. Tandem targeting/fusogenic peptides termed P51-DIV3W and P25-DIV3W were electrostatically complexed with non-targeting siRNA (siNT) at increasing N:P ratios to determine the minimum ratios needed to complex free siRNA. Various ratios of peptide-siRNA complexes were also treated with FBS and RNase A to determine whether the peptides protected siRNA from degradation. MTS assays were performed in SKBR3, BT474, and MCF10A cells to determine cytotoxicity using increasing peptide concentrations. Dynamic light scattering was performed using a Zetasizer to determine the size of the peptide-siRNA nanocomplexes. Results: Western Blot data of the three cell lines showed that CD44 protein was present in cancerous and normal cells, whereas HER2 protein was only present in SKBR3 and BT474 cells. Peptide-siRNA complexes for both P51-DIV3W and P25-DIV3W formed at a minimum N:P ratio of 20:1. Both peptide complexes protected siRNA from degradation in FBS and RNase A at N:P ratios ranging from 20:1 to 60:1. DLS results showed that P25-DIV3W-siRNA complexes had an average diameter of 200.23 nm and a zeta-potential of 24.7 mV. Conclusions: The data collected shows that the tandem peptides successfully complex with siNT and protect siNT from degradation. The peptide complexes formulate at a size that serves as an effective carrier for siRNA to breast cancer cells. Future work will focus on evaluating the ability of the tandem peptides to target HER2 and effectively silence CD44 in HER2+ breast cancer cells. Acknowledgements: This research was supported in part by the Dabo Swinney All In Foundation and Materials Assembly and Design Excellence in South Carolina (MADE in SC) under the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740. We also thank SC BioCRAFT, NIH award P30 GM131959, for the use of core equipment and facilities. Citation Format: James W. Kalogeros, Audreanna Miserendino, Angela Alexander-Bryant, Brian Booth. Tandem peptides targeting HER2 for delivery of CD44 siRNA into HER2+ breast cancer cells in vitro. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3979.